Download Comparison of the effects of macrolides, amoxicillin, ceftriaxone,

Comparison of the effects of macrolides, amoxicillin, ceftriaxone,
doxycycline, tobramycin and fluoroquinolones, on the production
of pneumolysin by Streptococcus pneumoniae in vitro
R. Anderson1,, H. C. Steel1, R. Cockeran1, A. von Gottberg2, L. de Gouveia2, K. P. Klugman2,3, T. J. Mitchell4 and
C. Feldman5
1
Medical Research Council (MRC) Unit for Inflammation and Immunity, Department of Immunology, Faculty of Health
Sciences, University of Pretoria, and Tshwane Academic Division of the National Health Laboratory Service, South Africa
2
MRC/NICD/WITS Respiratory and Meningeal Pathogens Research Unit, National Institute for Communicable Diseases,
Johannesburg, South Africa
3
Hubert Department of Global Health, Rollins School of Public Health, and Division of Infectious Diseases, School of
Medicine, Emory University, Atlanta, GA, USA
4
Division of Infection and Immunity, Glasgow Biomedical Research Centre, University of Glasgow, Scotland, UK
5
Division of Pulmonology, Department of Medicine, Johannesburg Hospital and University of the Witwatersrand,
Johannesburg, South Africa
Abstract
Objectives: To compare the effects of subinhibitory concentrations of amoxicillin, ceftriaxone,
azithromycin, clarithromycin, erythromycin, telithromycin, clindamycin, ciprofloxacin,
moxifloxacin, tobramycin and doxycycline on pneumolysin production by a macrolidesusceptible strain and two macrolide-resistant strains [erm(B) or mef(A)] of Streptococcus
pneumoniae. Methods: Pneumolysin was assayed using a functional procedure based on the
influx of Ca2+ into human neutrophils. Results: Only the macrolides/macrolide-like agents
caused significant attenuation of the production of pneumolysin, which was evident with all
three strains of the pneumococcus. Conclusions: Macrolides, at sub-MICs, but not other
classes of antibiotic, subvert the production of pneumolysin, even in the presence of (and
irrespective of the mechanism of) macrolide resistance in S. pneumoniae.
Introduction
We and others have reported that macrolide antibiotics, at therapeutically relevant
concentrations, inhibit the production of the pneumococcal toxin, pneumolysin, by macrolideresistant strains of Streptococcus pneumoniae in vitro and in vivo.1–3 Pneumolysin is believed
to cause bacteraemic disease by promoting extra-pulmonary dissemination of the
pneumococcus.4 Clarithromycin-mediated inhibition of the production of pneumolysin by
macrolide-resistant strains of the pneumococcus was evident at sub-MICs of this antimicrobial
agent and was independent of the type of macrolide resistance expressed (erm or mef
genes).3 However, relatively little is known about the comparative effects of different types of
macrolides and macrolide-like agents at sub-MICs on the production of pneumolysin by both
macrolide-susceptible and macrolide-resistant strains of S. pneumoniae, as well as the effects
on production of the toxin of other classes of antibiotics that may be used in the treatment of
pneumococcal infection.
In the current study, we have compared the effects of macrolides (clarithromycin,
erythromycin, azithromycin, telithromycin and clindamycin) with those of amoxicillin,
ceftriaxone, ciprofloxacin, moxifloxacin, tobramycin and doxycycline, all at a fixed final
concentration of 0.1 mg/L, on the production of pneumolysin by a macrolide-susceptible strain
of S. pneumoniae, as well as two resistant strains expressing either the erm or mef genes. The
concentration of 0.1 mg/L was either subinhibitory or close to the MIC value for most of the
tested antibiotics.
Materials and Methods
Antimicrobial agents
Pure substances of amoxicillin, clindamycin, doxycycline and tobramycin were purchased from
Sigma Chemical Co, St Louis, MO, USA, whereas the other antimicrobial agents
(azithromycin, clarithromycin, erythromycin, telithromycin, ceftriaxone, ciprofloxacin and
moxifloxacin) were kindly supplied by the relevant pharmaceutical manufacturers. All agents
were made to stock solutions of 1 g/L in distilled water.
Bacteria
One clinical macrolide-susceptible strain of S. pneumoniae (strain 172) and two macrolideresistant strains, 2507 and 521, which express the erm(B) and mef(A) genes, respectively, all
isolated in South Africa and known to be serotype 23F, were used in this study, and the
molecular/microbiological procedures used to confirm the identity of these strains are
described in detail elsewhere.3 Currently, 23F is an important clonal, clinical isolate that has
spread to many countries, and for which we had a well-characterized laboratory strain, as well
as genetically identical clinical isolates with varying degrees of macrolide resistance.3
Effects of the antimicrobial agents on pneumolysin production
To investigate the effects of the test antimicrobial agents on pneumolysin production,
uncomplicated by their inhibitory effects on bacterial proliferation, the macrolide-susceptible
and macrolide-resistant strains of S. pneumoniae were cultured in tryptone soy broth (TSB;
Biolab Diagnostics, Johannesburg, South Africa), for 6 h at 37°C in an atmosphere of 5% CO2,
after which they were harvested by centrifugation, then transferred to and washed in indicatorfree tissue culture medium RPMI 1640 [Highveld Biological (Pty) Ltd, Johannesburg, South
Africa]. Bacterial suspensions were then adjusted to give concentrations of 0.5–3 x 108 cfu/mL,
depending on the strain. The bacteria were incubated for 1 h at 37°C in an atmosphere of 5%
CO2 with the 11 different test antimicrobial agents at a fixed, final concentration of 0.1 mg/L.
Following the 1 h incubation period, bovine serum albumin (5 g/L final; Sigma Chemical Co)
was added to each tube, followed by a further incubation period of 16 h, after which
pneumolysin was assayed both in the bacteria-free supernatants and in the lysates
(sonicates), as described below. Pneumolysin in supernatants/sonicates was also measured
at the outset, immediately before exposure of the bacteria to the antibiotics.
Pneumolysin assay
Pneumolysin in the bacteria-free supernatants and sonicates was measured using a fura-2/AM
(Sigma Chemical Co)-based spectrofluorimetric procedure that detects toxin-mediated influx of
Ca2+ into isolated human neutrophils, as described in detail elsewhere.3
Effects of clarithromycin on bacterial protein synthesis
Each of the three strains of the pneumococcus was cultured in the absence or presence of 0.1
mg/L clarithromycin, as mentioned earlier, in RPMI 1640 supplemented with 0.5 mCi/L of a
radiolabelled amino acid mixture (L-amino acid mixture 14C[U], 37 MBq, Du Pont-NEN
Products, Boston, MA, USA) and incubated at 37°C/5% CO2. After 6 h of incubation, the
bacteria were pelleted by centrifugation and washed, followed by the addition of warm 5%
trichloroacetic acid to lyse the bacteria and release proteins. Radioactivity in the lysates was
measured using liquid scintillation spectrometry.
Statistical analysis
The results of each series of experiments are presented as mean values±SEMs. Levels of
statistical significance were calculated using the Student's t-test (unpaired t statistic).
Results
MIC values
MIC values of each strain are shown in Table 1. As expected, strains 2507 and 521 were
resistant to azithromycin, clarithromycin and erythromycin, whereas strain 2507 was resistant
to clindamycin, and both strains were susceptible to telithromycin. Strain 172 was susceptible
to all macrolides/macrolide-like agents. All three test strains of S. pneumoniae were
susceptible to the remaining antibiotics. For all of the strains tested, 0.1 mg/L represented
either a sub-MIC or was close to the MIC value; doxycycline and telithromycin were the
exceptions with MIC values for each strain being higher and lower, respectively, than 0.1
mg/L. Strain 521 was particularly susceptible to amoxicillin, whereas strain 2507 was resistant
to clindamycin.
Table 1. MICs of the test antimicrobial agents for the macrolide-susceptible and macrolide-resistant strains of S.
pneumoniae
MIC (mg/L)
Antimicrobial agents
172a
2507b
521b
Azithromycin
0.5
>256
8
Clarithromycin
0.064
>256
2
Erythromycin
0.094
>256
4
Telithromycin
0.015
0.03
0.015
Clindamycin
0.06
>256
0.06
Amoxicillin
0.75
0.25
0.023
Ceftriaxone
1
0.5
0.06
Ciprofloxacin
0.19
0.38
0.38
Moxifloxacin
0.05
0.094
0.064
Tobramycin
0.125
0.19
0.125
Doxycycline
4
4
8
Pneumolysin
The effects of the test antibiotics on the production of total (intracellular + extracellular)
pneumolysin by the macrolide-susceptible and two macrolide-resistant strains of S.
pneumoniae are shown in Figure 1. Exposure of all three strains to clarithromycin,
erythromycin, telithromycin and clindamycin was accompanied by significant decreases in the
levels of pneumolysin. The effects of azithromycin, although similar to those of the other
macrolides/macrolide-like agents, were of a lesser magnitude, and, in several instances, did
not achieve statistical significance. With the exception of amoxicillin, which suppressed the
production of pneumolysin by strain 521, probably due to the high level of sensitivity of strain
521 to this antibiotic, none of the other antimicrobial agents affected the production of the
toxin. The inhibitory effects of the antibiotics on the concentration of extracellular and total
pneumolysin were comparable (data not shown).
Figure 1. Effects of the test antimicrobial agents on the production of pneumolysin by (a) strain 172 (macrolidesusceptible) and strains (b) 2507 and (c) 521 (macrolide-resistant, erm and mef, respectively) of S. pneumoniae.
The results of four experiments are expressed as the mean values ± SEM for total pneumolysin; *P < 0.05 for
comparison with the corresponding antibiotic-free control systems.
Because of the relatively high MIC values of doxycycline for all three strains of the
pneumococcus, we also investigated the effect of this agent at a concentration of 2 mg/L on
toxin production. At this higher concentration, doxycycline caused significant inhibition of
pneumolysin production by all three strains of the pneumococcus; the mean percentages of
inhibition (±SEMs) are 63 ± 5, 34 ± 5 and 67 ± 3 (P < 0.05 for each value) for strains 172, 521
and 2507, respectively.
The numbers of viable bacteria (cfu) were 2.1 ± 0.64, 1.5 ± 0.4 and 1.2 ± 0.5 x 108 cfu/mL for
strains 172, 2507 and 521, respectively, at the outset, whereas the corresponding values after
16 h of incubation in RPMI were 1.3 ± 0.8, 2.3 ± 0.5 and 0.9 ± 0.2 x 106 cfu/mL. With the
exception of significantly (P < 0.05) decreased numbers of viable bacteria following the 16 h
exposure of strain 521 to amoxicillin (0.9 ± 0.2 x 106 versus 0.2 ± 0.1 x 106 cfu/mL), there were
no significant differences between the numbers of viable bacteria in systems without and with
the antibiotics after 16 h of incubation.
Protein synthesis
The effects of clarithromycin (0.1 mg/L) on bacterial protein synthesis are shown in Figure 2.
Significant (P < 0.05) inhibition of protein synthesis was observed with all three strains of the
pneumococcus.
Figure 2. Effects of clarithromycin (0.1 mg/L) on protein synthesis by strain 172 (macrolide-susceptible)
and strains 2507 and 521 (macrolide-resistant, erm and mef, respectively) of S. pneumoniae. The results
of three different experiments with three to five replicates for each system are expressed as the mean
values ± SEM for total protein synthesis; *P < 0.05 for comparison with the corresponding clarithromycinfree control systems.
Discussion
Only the macrolides and macrolide-like antibiotics were found to inhibit the production of
pneumolysin by all three strains of the pneumococcus, with clarithromycin, erythromycin,
telithromycin and clindamycin exhibiting comparable activities, whereas azithromycin was
generally somewhat less active. Importantly, macrolides, at the concentration used in the
current study, do not interfere with the pneumolysin assay system.3 Neither doxycycline nor
tobramycin, both inhibitors of bacterial protein synthesis, affected the production of
pneumolysin by any of the test strains of the pneumococcus. However, increasing the
concentration of doxycycline to 2 mg/L resulted in significant inhibition of the synthesis of
pneumolysin by all three strains of the pneumococcus.
Inhibition of protein synthesis, as opposed to possible non-ribosomal mechanisms of
antimicrobial activity, appears to be involved in the macrolide-mediated inhibition of synthesis
of pneumolysin by macrolide-resistant strains of the pneumococcus. This contention is based
on the observation that clarithromycin (0.1 mg/mL) inhibited protein synthesis by macrolideresistant strains of the pneumococcus, albeit to a lesser extent than that observed with the
macrolide-susceptible strain. Macrolides and clindamycin, at subinhibitory concentrations,
have also been reported to interfere with several virulence-related activities of Pseudomonas
aeruginosa, including biofilm formation, twitching motility and quorum sensing.5–8 Interestingly,
these unusual effects of macrolides on P. aeruginosa, as well as those on pneumolysin
production by macrolide-resistant strains of the pneumococcus described in the current study,
are unlikely to be detected by conventional assays of in vitro antibiotic susceptibility testing.
In comparison with the other classes of antibiotic tested, macrolides at sub-MICs effectively
antagonize the production of pneumolysin by both macrolide-susceptible and macrolideresistant strains of the pneumococcus, compatible with a role for these agents as adjuncts to
ß-lactams in the treatment of severe pneumococcal disease.9,10
Transparency declarations
C. F. has acted on the advisory board of pharmaceutical companies manufacturing and/or
marketing antibiotics (MSD, Abbott, Pfizer and Sanofi-Aventis) and has received honoraria for
lectures from pharmaceutical companies manufacturing and/or marketing antibiotics (Abbott,
MSD, GlaxoSmithKline, Pfizer and Sanofi-Aventis) and has also received assistance for
congress attendance from pharmaceutical companies manufacturing antibiotics (Abbott,
GlaxoSmithKline and Sanofi-Aventis). K. P. K. is in receipt of research funding from Bayer and
Sanofi-Aventis and has received consultant fees from Bayer, Sanofi-Aventis and
GlaxoSmithKline in the last year.
Funding
The study was supported in part by a grant awarded to R. C. by the National Health
Laboratory Service of South Africa Research Trust. R. A. has received a research grant from
Abbott Laboratories in partial support of the current study.
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